Gold nanocarriers for transport of oligonucleotides across brain endothelial cells

Treatment of diseases that affect the CNS by gene therapy requires delivery of oligonucleotides to target cells within the brain. As the blood brain barrier prevents movement of large biomolecules, current approaches involve direct injection of the oligonucleotides, which is invasive and may have only a localised effect. The aim of this study was to investigate the potential of 2 nm galactose-coated gold nanoparticles (NP-Gal) as a delivery system of oligonucleotides across brain endothelium. DNA oligonucleotides of different types were attached to NP-Gal by the place exchange reaction and were characterised by EMSA (electrophoretic mobility shift assay). Several nanoparticle formulations were created, with single- or double-stranded (20nt or 40nt) DNA oligonucleotides, or with different amounts of DNA attached to the carriers. These nanocarriers were applied to transwell cultures of human brain endothelium in vitro (hCMEC/D3 cell-line) or to a 3D-hydrogel model of the blood-brain barrier including astrocytes. Transfer rates were measured by quantitative electron microscopy for the nanoparticles and qPCR for DNA. Despite the increase in nanoparticle size caused by attachment of oligonucleotides to the NP-Gal carrier, the rates of endocytosis and transcytosis of nanoparticles were both considerably increased when they carried an oligonucleotide cargo. Carriers with 40nt dsDNA were most efficient, accumulating in vesicles, in the cytosol and beneath the basal membrane of the endothelium. The oligonucleotide cargo remained attached to the nanocarriers during transcytosis and the transport rate across the endothelial cells was increased at least 50fold compared with free DNA. The nanoparticles entered the extracellular matrix and were taken up by the astrocytes in biologically functional amounts. Attachment of DNA confers a strong negative charge to the nanoparticles which may explain the enhanced binding to the endothelium and transcytosis by both vesicular transport and the transmembrane/cytosol pathway. These gold nanoparticles have the potential to transport therapeutic amounts of nucleic acids into the CNS

The use of vibrational spectroscopy to study the pathogenesis multiple sclerosis and other neurological conditions

Spectroscopy techniques are valuable tools in biomedical research and have been used extensively in the study of disease. However, neurological conditions such as multiple sclerosis (MS) have received little attention and the available spectroscopy studies are limited, both in overall numbers of patients studied and the disease samples considered. MS is a complex immune-mediated disease, with variable clinical courses and limited therapeutic options. This review aims to summarize current literature in the area, demonstrating how spectroscopy techniques can provide valuable information to inform and advance research into the most common neurological condition affecting young adults

Transport of Gold Nanoparticles by Vascular Endothelium from Different Human Tissues

The selective entry of nanoparticles into target tissues is the key factor which determines their tissue distribution. Entry is primarily controlled by microvascular endothelial cells, which have tissue-specific properties. This study investigated the cellular properties involved in selective transport of gold nanoparticles (<5 nm) coated with PEG-amine/galactose in two different human vascular endothelia. Kidney endothelium (ciGENC) showed higher uptake of these nanoparticles than brain endothelium (hCMEC/D3), reflecting their biodistribution in vivo. Nanoparticle uptake and subcellular localisation was quantified by transmission electron microscopy. The rate of internalisation was approximately 4x higher in kidney endothelium than brain endothelium. Vesicular endocytosis was approximately 4x greater than cytosolic uptake in both cell types, and endocytosis was blocked by metabolic inhibition, whereas cytosolic uptake was energy-independent. The cellular basis for the different rates of internalisation was investigated. Morphologically, both endothelia had similar profiles of vesicles and cell volumes. However, the rate of endocytosis was higher in kidney endothelium. Moreover, the glycocalyces of the endothelia differed, as determined by lectin-binding, and partial removal of the glycocalyx reduced nanoparticle uptake by kidney endothelium, but not brain endothelium. This study identifies tissue-specific properties of vascular endothelium that affects their interaction with nanoparticles and rate of transpor

Cytokine-induced changes in the gene expression profile of a human cerebral microvascular endothelial cell-line, hCMEC/D3.

Background: The human cerebral microvascular endothelial cell line, hCMEC/D3, has been used extensively to model the blood–brain barrier (BBB) in vitro. Recently, we reported that cytokine-treatment induced loss of brain endothelial barrier properties. In this study, we further determined the gene expression pattern of hCMEC/D3 cells in response to activation with TNFα and IFNγ. Findings: Using a microarray approach, we observed that expression of genes involved in the control of barrier permeability, including inter-brain endothelial junctions (e.g. claudin-5, MARVELD-2), integrin-focal adhesions complexes (e.g. integrin β1, ELMO-1) and transporter systems (e.g. ABCB1, SLC2A1), are altered by pro-inflammatory cytokines. Conclusions: Our study shows that previously-described cytokine-induced changes in the pattern of gene expression of endothelium are reproduced in hCMEC/D3 cells, suggesting that this model is suitable to study inflammation at the BBB, while at the same time it has provided insights into novel key molecular processes that are altered in brain endothelium during neuroinflammation, such as modulation of cell-to-matrix contacts

Autologous hematopoietic cell transplantation in multiple sclerosis.

INTRODUCTION: Autologous haematopoietic cell transplantation (AHCT) is an evolving treatment avenue in multiple sclerosis (MS), which may be highly effective in controlling disease activity and improving disability. However, AHCT is associated with intrinsic toxicities and risks compared with conventional therapies. With growing experience in patient selection and treatment delivery, AHCT is increasingly considered an option in patients with aggressive disease that's responding poorly to disease modifying therapy. AREAS COVERED: This article provides an introduction to AHCT and looks at its development as a treatment for MS over the last 20 years. It also highlights potential mechanisms of action, patient selection, and future trends for this treatment approach. EXPERT OPINION: Currently published data suggest that AHCT's use is associated with significant reduction in MS disease activity and marked improvement in disability when used in patients with highly active relapsing remitting disease. Its long term safety and efficacy have not been fully evaluated but as increasing clinical trial data are published, its use is likely to grow. Further randomised controlled studies are needed to compare AHCT with standard disease modifying therapies and to optimise transplant regimens. Mechanistic studies may provide potential markers for response and a better understanding of disease pathogenesis

Cerebrospinal fluid dynamics modulation by diet and cytokines in rats.

Background Idiopathic intracranial hypertension (IIH) is a neurological disorder characterised by raised cerebrospinal fluid (CSF) pressure in the absence of any intracranial pathology. IIH mainly affects women with obesity between the ages of 15 and 45. Two possible mechanisms that could explain the increased CSF pressure in IIH are excessive CSF production by the choroid plexus (CP) epithelium or impaired CSF drainage from the brain. However, the molecular mechanisms controlling these mechanisms in IIH remain to be determined. Methods In vivo ventriculo-cisternal perfusion (VCP) and variable rate infusion (VRI) techniques were used to assess changes in rates of CSF secretion and resistance to CSF drainage in female and male Wistar rats fed either a control (C) or high-fat (HF) diet (under anaesthesia with 20 μl/100 g medetomidine, 50 μl/100 g ketamine i.p). In addition, CSF secretion and drainage were assessed in female rats following treatment with inflammatory mediators known to be elevated in the CSF of IIH patients: C-C motif chemokine ligand 2 (CCL2), interleukin (IL)-17 (IL-17), IL-6, IL-1β, tumour necrosis factor-α (TNF-α), as well as glucocorticoid hydrocortisone (HC). Results Female rats fed the HF diet had greater CSF secretion compared to those on control diet (3.18 ± 0.12 μl/min HF, 1.49 ± 0.15 μl/min control). Increased CSF secretion was seen in both groups following HC treatment (by 132% in controls and 114% in HF) but only in control rats following TNF-α treatment (137% increase). The resistance to CSF drainage was not different between control and HF fed female rats (6.13 ± 0.44 mmH O min/μl controls, and 7.09 ± 0.26 mmH O min/μl HF). and when treated with CCL2, both groups displayed an increase in resistance to CSF drainage of 141% (controls) and 139% (HF) indicating lower levels of CSF drainage. Conclusions Weight loss and therapies targeting HC, TNF-α and CCL2, whether separately or in combination, may be beneficial to modulate rates of CSF secretion and/or resistance to CSF drainage pathways, both factors likely contributing to the raised intracranial pressure (ICP) observed in female IIH patients with obesity.</p

Multiple brain networks support processing speed abilities of patients with multiple sclerosis

Objectives: Many people affected by multiple sclerosis (MS) experience cognitive impairment, especially decreases in information processing speed (PS). Neural disconnection is thought to represent the neural marker of this symptom, although the role played by alterations of specific functional brain networks still remains unclear. The aim is to investigate and compare patterns of association between PS-demanding cognitive performance and functional connectivity across two MS phenotypes. Methods: Forty patients with relapsing-remitting MS (RRMS) and 25 with secondary progressive MS (SPMS) had neuropsychological and MRI assessments. Multiple regression models were used to investigate the relationship between performance on tests of visuomotor and verbal PS, and on the verbal fluency tests, and functional connectivity of four cognitive networks, i.e. left and right frontoparietal, salience and default-mode, and two control networks, i.e. visual and sensorimotor. Results: Patients with SPMS were older and had longer disease history than patients with RRMS and presented with worse overall clinical conditions: higher disease severity, total lesion volume, and cognitive impairment rates. However, in both patient samples, cognitive performance across tests was negatively correlated with functional connectivity of the salience and default-mode networks, and positively with connectivity of the left frontoparietal network. Only the visuomotor PS scores of the RRMS group were also associated with connectivity of the sensorimotor network. Conclusions: PS-demanding cognitive performance in patients with MS appears mainly associated with strength of functional connectivity of frontal networks involved in the evaluation and manipulation of information, as well as the default mode network. These results are in line with the hypothesis that multiple neural networks are needed to support normal cognitive performance across MS phenotypes. However, different PS measures showed partially different patterns of association with functional connectivity. Therefore, further investigations are needed to clarify the contribution of inter-network communication to specific cognitive deficits due to MS

Qualitative investigation of exercise perceptions and experiences in people with multiple sclerosis before, during, and after participation in a personally tailored exercise program

Objective To undertake a qualitative investigation of exercise perceptions and experiences in people with multiple sclerosis (PwMS) before, during, and after participation in a personally tail ored program designed to promote long-term maintenance of self-directed exercise. Design Focus groups and semistructured telephone interviews. Setting University exercise science department close to the recruiting hospital. Participants PwMS (N=33; mean age ± SD, 47.6±7.9y). Interventions Participants were recruited after participation in a randomized controlled exercise trial; all had been allocated to a 12-week exercise program comprising supervised and self-directed exercise sessions. Main Outcome Measures Exercise perceptions and experiences before, during, and after participation in the program. Results Four themes emerged from the analysis: (1) the transition to inactivity; (2) lack of knowledge and confidence; (3) positive exercise experiences; and (4) perspectives on exercise adherence. Conclusions Lack of confidence and exercise knowledge, coupled with negative perceptions about physical capabilities after an MS diagnosis, are clear barriers to exercise participation in PwMS. These issues are not being adequately addressed as part of the health care pathway or in community settings. Perceptions of improved posture, ability to overcome everyday difficulties, acute mood enhancements during and after exercise, and increased opportunities for social interaction were among the reported benefits of exercise participation. Despite the provision of a personally tailored exercise plan and use of cognitive behavioral strategies, self-directed exercise continued to present challenges to PwMS, and the importance of seeking cost-effective ways to maintain motivational support was implicit in participant responses

Listening to the patients' voice: a conceptual framework of the walking experience

BACKGROUND: walking is crucial for an active and healthy ageing, but the perspectives of individuals living with walking impairment are still poorly understood. OBJECTIVES: to identify and synthesise evidence describing walking as experienced by adults living with mobility-impairing health conditions and to propose an empirical conceptual framework of walking experience. METHODS: we performed a systematic review and meta-ethnography of qualitative evidence, searching seven electronic databases for records that explored personal experiences of walking in individuals living with conditions of diverse aetiology. Conditions included Parkinson's disease, multiple sclerosis, chronic obstructive pulmonary disease, hip fracture, heart failure, frailty and sarcopenia. Data were extracted, critically appraised using the NICE quality checklist and synthesised using standardised best practices. RESULTS: from 2,552 unique records, 117 were eligible. Walking experience was similar across conditions and described by seven themes: (i) becoming aware of the personal walking experience, (ii) the walking experience as a link between individuals' activities and sense of self, (iii) the physical walking experience, (iv) the mental and emotional walking experience, (v) the social walking experience, (vi) the context of the walking experience and (vii) behavioural and attitudinal adaptations resulting from the walking experience. We propose a novel conceptual framework that visually represents the walking experience, informed by the interplay between these themes. CONCLUSION: a multi-faceted and dynamic experience of walking was common across health conditions. Our conceptual framework of the walking experience provides a novel theoretical structure for patient-centred clinical practice, research and public health